Calcium (Ca2+) is a multifunctional signalling ion that regulates a wide range of cellular functions ranging from short-term responses, such as contraction and secretion, to longer-term control of transcription, cell division and cell death (Jeremy T. Smyth, et al.; 2010. J. Cell. Mol. Med. Vol 14, No 10, pp. 2337-2349; Lewis Richard S. 2011 Cold Spring Harb Perspect Biol; 3:a003970). The very low concentration of Ca2+ in the cytosol together with the massive gradient across membranes (Ca2+ is 105 times more abundant in organelles and in the extracellular medium) became thereafter a great opportunity to use this ion as a specific second messenger. A Ca2+-signal encodes a message through its amplitude, the duration of its rise, the frequency of its rises and the exact spatial localization in the cell. It has grown to such specialization that hundreds of proteins govern this process and each cell type has a unique set of proteins that are chosen for particular tasks, the “Ca2+ toolkit”, as defined by one of the fathers of modern Ca2+ signalling (Berridge M J, Bootman M D, Roderick H L. 2003. Nature Reviews. Molecular Cell Biology 4:517-529). As mentioned above, high concentrations of Ca2+ are present in intracellular organelles (with particular reference to the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR)) and in the extracellular space and Ca2+-fluxes occur through channels located on the plasma membrane or on the membrane of intracellular organelles. Given that Ca2+-pumps and exchangers are located on both membranes to extrude Ca2+ from the cytosol, it would be expected that the intracellular organelle pool would be soon depleted. Among these pathways, store-operated-Ca2+ entry (SOCE), so named for its regulation by the free Ca2+ concentration ([Ca2+]) of the ER Ca2+ stores, is a widespread Ca2+ entry mechanism in animal cells that delivers Ca2+ to refill ER stores and evoke cellular Ca2+ signals (Putney J W. 2011. Frontiers in Bioscience (Scholar Edition) 3:980-984).
Store-operated Ca2+ entry is associated with the electrophysiological current ICRAC, first described by Hoth and Penner (Hoth M, Penner R. 1992. Nature 355:353-356). The exact molecular mechanisms behind this phenomenon have been elusive for a number of years, but it is now thought that the principal components of the machinery are the Ca2+-release-activated-Ca2+ (CRAC) channels. CRAC channels are assembled from two fundamental protein complexes: Orai proteins, that form the ion channel pore, and the stromal interaction molecule (STIM) proteins, which function as ER calcium sensor and activators of the CRAC channels (Berna-Erro A, et al. Redondo P C, Rosado J A. 2012. Medicine and Biology 740:349-382; Soboloff J, Rothberg B S, Madesh M, Gill D L. 2012. Nature Reviews. Molecular Cell Biology 13:549-565.; Lacruz R S, Feske S. 2015. Annals of the New York Academy of Sciences 1356:45-79.). Moreover, it is important to stress that other crucial proteins participate in the SOCE process, including TRPC channels (Ong H L, Ambudkar I S. 2015. Cell Calcium 58:376-386).
STIM proteins are single-span membrane proteins, highly conserved across species. Two members of the family have been described, STIM1 and STIM2, of which the former appears more expressed. Roos et al. (2005; J Cell Biol.; 169(3):435-45.) using a limited RNAi screen of Drosophila S2 cells identified Drosophila STIM as having a fundamental role in SOCE activation, and a similar conclusion was reached almost concurrently for human STIM1 and STIM2 in a HeLa cell screen (Jeremy T. Smyth, et al.; 2010. J. Cell. Mol. Med. Vol 14, No 10, pp. 2337-2349, Lewis Richard S. 2011 Cold Spring Harb Perspect Biol; 3:a003970). STIM1 was identified as a Ca2+ sensor for SOCE since it is specialized for responding to significant changes in ER Ca2+ signals. STIM1 localization is crucial to its role SOCE: when Ca2+ stores are full STIM1 is localized in tubular structures throughout the ER membrane, but when stores are depleted it moves to punctate structures at site where the ER is directly apposed to the plasma membrane. This re-localization of STIM1 within the ER towards the plasma membrane allows the direct or indirect interaction and activation of Orai channels. Orai channels reside on the plasma membrane and three members of the family (Orai1, Orai2, and Orai3) have been described, with Orai1 being the most abundant and closely connected to the ICRAC (Jeremy T. Smyth, et al.; 2010. J. Cell. Mol. Med. Vol 14, No 10, pp. 2337-2349; Lewis Richard S. 2011 Cold Spring Harb Perspect Biol; 3:a003970; Feske S, et al; 2005 J Exp Med.; 202(5):651-62; Nature; 11; 441(7090):179-85.).
CRAC currents were initially identified in lymphocyte and mast cells, and simultaneously characterized in different cell lines such as DT40 B cells, hepatocytes, dendritic, megakaryotic and Madin-Darby canine kidney cells. In lymphocyte and mast cells, the activation through antigen or Fc receptor initiates the release of Ca2+ ion from intracellular stores caused by the second messenger inositol (1,4,5)-triphosphate (IP3), that leads to Ca2+ ion influx through CRAC channels in the plasma membrane.
CRAC channels also mediate crucial function from secretion to gene expression and cell growth and form a process essential for the activation of adaptive immune response. It has been demonstrated that Ca2+ oscillations triggered through stimulation of the T-cell antigen receptor (TCR) involved only the influx pathway of the store operated CRAC channel. Therefore, Ca2+ ion influx mediated by the store operated CRAC channel is fundamental in lymphocyte activation (Anant B. Parekh and James W. Putney Jr.; 2005; Physiol Rev 85: 757-810.; Hogan G. p., et al; 2010; Annu. Rev. Immunol. 28:491-533; Patrick G Hogan and Anjana Rao; 2015; Biochem Biophys Res Commun. 24; 460(1): 40-49.; Feske S, Okamura H, Hogan P G, Rao A. 2003; Biochem Biophys Res Commun.; 311(4):1117-32.). Conversely, the store-operated Ca2+ currents identified in endothelial cells, smooth muscle, epidermal cells and prostate cancer cells lines show altered biophysical characteristic suggesting a different molecular origin. These evidences demonstrate that intracellular Ca2+ plays an important role in different cellular functions, and its concentration by Ca2+ influx through Ca2+ channels on the plasma membrane and ER.
Furthermore, the importance of CRAC channels for human health is underlined by an increasing list of genetic studies that have identified that patients who bear loss- or gain-of-function STIM1/Orai1 mutations suffer from severe health issues, including muscle defects, immunodeficiency, autoimmunity and bleeding disorders (Feske S. 2010; European Journal of Physiology 460:417-435). Regarding loss-of-function mutations, at least three unrelated families have been described that, due to different mutations, including frame-shifts, do not express Orai1 on the plasma membrane of T-lymphocytes, lack store-operated Ca2+-entry and are thereby unable to activate T-lymphocytes (Feske S, Muiller J M, Graf D, Kroczek R A, Drager R, Niemeyer C, Baeuerle P A, Peter H H, Schlesier M. 1996. European Journal of Immunology 26:2119-2126.; McCarl C A, et al.; 2009. J Allergy Clin Immunol.; 124(6):1311-1318.e7.) Notably, families with STIM1 mutations that lead to no expression of the protein have been reported and are characterized by a T-cell immunodeficiency (Picard C, et al.; 2009, N Engl J Med. 7; 360(19):1971-80.; Byun M, et al.; 2010. The Journal of Experimental Medicine. 207:2307-2312.; Fuchs S, et al.; 2012. Journal of Immunology (Baltimore, Md.: 1950) 188:1523-1533). Last, while immunodeficiency is the hallmark of the disease, these patients also display lymphoproliferative diseases, autoimmunity, congenital myopathy, anhydrosis, dental enamel, and an impairment in thrombus formation due to a defect in platelet activation. While some mutations give rise to a decreased activity that might be potentiated pharmacologically, most mutations yield a significant decrease in protein expression and therefore pharmacological approaches might be indicated also for these disorders. Currently, the loss-of-function mutations of STIM1 and Orai1 reported in the literature are the following: p.P165Q, p.R429C, p.R426C, p.E128RfsX9 for STIM1, and p.R91W, p.A103E, p.L194P, p.A88SfsX25 and p.H165PfsX1 for Orai1.
Gain-of-function mutations of STIM1 or Orai1 affect primarily skeletal muscles and platelets. Patients with STIM1 or Orai1 gain-of-function mutations exhibit a wide and likely continuous spectrum of symptoms that affect multiple organs and are different in intensity, progression and in age of onset. Yet, skeletal muscle and platelets appear the main systems affected. Three separate disorders (tubular aggregate myopathy, Stormorken syndrome and York platelet syndrome) are described in the literature and can be reconducted to mutations in one of these two proteins (Lacruz R S, Feske S. 2015. Annals of the New York Academy of Sciences 1356:45-79). Tubular aggregate myopathy (TAM) can be re-conducted to gain-of-function mutations in either STIM1 and Orai1, and is clinically characterized by variable combinations of myalgias, cramps and muscle stiffness, with or without weakness with a predominantly proximal distribution (Bihm J, et al. 2013. American Journal of Human Genetics 92:271-278; Nesin V, et al. 2014. Proceedings of the National Academy of Sciences of the United States of America 111:4197-4202; Endo Y, et al. 2015. Human Molecular Genetics 24:637-648.) and by the presence of tubular aggregates, which are regular arrays of tubules derived from the sarcoplasmic reticulum, on muscle pathology (Schiaffino S. 2012. Neuromuscul Disord 22:199-207.). Stormorken syndrome (Stormorken H, et al. 1995. Thromb Haemost 74:1244-1251) associates to the myopathic signs, but may also include mild bleeding tendency due to platelet dysfunction, thrombocytopenia, anemia, asplenia, congenital miosis, ichthyosis, and may also include headache or recurrent stroke-like episodes (Misceo D, et al 2014. Human Mutation 35:556-564.). Also in this case, both STIM1 and Orai1 (Nesin V, et al. 2014. Proceedings of the National Academy of Sciences of the United States of America 111:4197-4202) mutations have been associated with the syndrome. Last, the York platelet syndrome, recently described in the United States, sees blood dyscrasias as the main phenotype (Markello T et al. Molecular Genetics and Metabolism 114:474-482.) and has, so far, only been associated to STIM1 mutations. Currently, the gain-of-function mutations of STIM1 and Orai1 reported in the literature are the following: p.F108I, p.I115F, p.H109R, p.H72Q, p.N80T, p.G81D, p.D84G, p.L96V, p.F108L, p.H109N, p.R304W, p.R304G for STIM1; and p.S97C, p.G98S, p.L138F, p.P245L for Orai1.
Briefly, mutations of STIM1 mostly reside in the EF-hand Ca2+-binding motifs, most likely modifying the affinity for Ca2+ ions of the protein, with the single exception of a mutation located in the cytosolic side of the protein on a coil-coiled domain that is likely to affect dimerization/oligomerization of STIM1, a likely trigger of Orai1 channel opening. The mutations of Orai1 are located in the trans-membrane domains in positions that might lead to the assumption that they participate in the channel lining.
All these data suggest that SOCE modulators would be useful for the treatment of diseases caused by an abnormal SOCE. A key limitation in the study of SOCE and its physio- and pathophysiological role is the lack of potent and selective modulators. Some agents are available (Sweeney Z K, Minatti A, Button D C, Patrick S. 2009. ChemMedChem 4:706-718), but they share common drawbacks in terms of lacking potency and/or specificity over CRAC channels. Lanthanides (Ln3+) are nonselective cation channel blockers, while 2-aminoethoxydiphenylborate (2-APB), a repurposed inositol triphosphate receptor inhibitor, is a nonspecific blocker, which potentiates ICRAC at low micromolar concentrations, while inhibiting ICRAC at higher concentrations (Diver J M, et al. 2001. Cell Calcium 30:323-329). Carboxyamido-triazole (CAI) is a synthetic small molecule inhibitor of non-voltage-gated Ca2+ channels that has entered Phase I, II and III clinical trials, both as single cytostatic agent and in combination with cytotoxic therapies. CAI is, however, a nonspecific agent that may target cellular pathways other than non-voltage-gated Ca2+ channels (Lodola F, et al. 2012. PloS One 7:e42541.Lodola et al., 2012). While small molecules used in the past (2-APB, CAI) were found to be unselective, second generation modulators should block CRAC channels with a certain degree of selectivity. Synta66, a compound developed by GSK, inhibits ICRAC with an IC50 of 0.3 μM (Di Sabatino A. 2009. Journal of Immunology (Baltimore, Md.: 1950) 183:3454-3462), while Hoffmann-La Roche has recently reported the discovery of RO2959, a potent CRAC channel inhibitor with IC50 values of about 200 nM (Chen G, et al. 2013. Molecular Immunology 54:355-367). To achieve these IC50 values, however, this molecule has to be pre-incubated with cells for 30 to 60 minutes suggesting that it may act on Orai1 indirectly. Very recently, Dolmetsch et al. have screened a small-molecule microarray through an innovative approach that makes use of minimal functional domains and discovered AnCoA4, an isoflavone able to bind to and inhibit Orai1 at submicromolar concentrations (Sadaghiani A M, et al. 2014. Chemistry & Biology 21:1278-1292), but no data about its specificity are available.
Much interest has been directed at a series of 3,5-bistrifluoromethyl pyrazole derivatives, referred to as BTPs and disclosed by Abbott in 2000 (Djuric S W, et al. 2000. Journal of Medicinal Chemistry 43:2975-2981.; US 20010044445; US 20010044445). Specifically, BTP2 (Pyr2, YM-58483) is a potent inhibitor, but has pleiotropic effects on both Orai and TRPC channels (Takezawa R, et al. 2006. Molecular Pharmacology 69:1413-1420). Subsequently, other pyrazoles, identified as Pyr, have been reported. Pyr3, a previously suggested selective inhibitor of TRPC3 (Kiyonaka S, et al. 2009. Proceedings of the National Academy of Sciences of the United States of America 106:5400-5405; Glasnov T. N. et al., ChemMedChem, 2009, 4:1816-1818), was shown to inhibit both TRPC3- and Orai1 mediated Ca2+ entry (Schleifer H, et al. 2012. British Journal of Pharmacology 167:1712-1722). By contrast, two compounds, Pyr6 and Pyr10, are able to distinguish to a certain degree between Orai and TRPC-mediated Ca2+ entry (Schleifer H, et al. 2012. British Journal of Pharmacology 167:1712-1722). Most of these reported compounds display a pyrazole ring with at 1-position an arylamide moiety. Similar compounds are described in WO2006115140 by Astellas Pharma (Yonetoku Y. et al. Bioorg. Med. Chem. 2006, 14, 4750-4760; 2008, 16, 9457-9466; 2006, 14, 5370-5383), where pyrazolic CRAC inhibitors are reported to be useful in bowel diseases. Other patent publications relating to similar CRAC channel modulators include the applications by Icozen Therapeutics and Rhizen Pharmaceuticals, WO 2011042797 and WO2011042798, where the reported inhibitors are pyrazole derivatives in which a substituted pyrazole is bound to a phenyl or pyridine group carrying an inverse amide in para-position. Bioisosteric replacement of the arylamide moiety with a fused heterocycle scaffold led to a series of pyrazolic modulators described by Hoffmann-La Roche in the applications WO2013050270, WO2013050341, US20130158066, US20130158049 and US20130158040. GlaxoSmithKline published two separate patent applications in 2010: the first (WO2010122088) comprises both pyrazole and triazole carboxamides, while the second (WO2010122089) focuses exclusively on N-pyrazolyl carboxamides. The amide bond is reversed in WO2010122088 as compared with WO2010122089. Calcimedica reported pyrazole compounds as CRAC channels inhibitors in WO2011139765, US2011263612, WO2010048559 and WO2009076454.
Despite all the reported applications, there remains an unmet and dire need for small molecule modulators having specificity towards Stim1 and/or Orai1 in order to regulate activity of CRAC channels, particularly for the treatment of diseases and disorders associated with SOCE.